The cholinesterase-like domain of thyroglobulin functions as an intramolecular chaperone
Jaemin Lee, Bruno Di Jeso, Peter Arvan
Jaemin Lee, Bruno Di Jeso, Peter Arvan
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Research Article Endocrinology

The cholinesterase-like domain of thyroglobulin functions as an intramolecular chaperone

Abstract

Thyroid hormonogenesis requires secretion of thyroglobulin, a protein comprising Cys-rich regions I, II, and III (referred to collectively as region I-II-III) followed by a cholinesterase-like (ChEL) domain. Secretion of mature thyroglobulin requires extensive folding and glycosylation in the ER. Multiple reports have linked mutations in the ChEL domain to congenital hypothyroidism in humans and rodents; these mutations block thyroglobulin from exiting the ER and induce ER stress. We report that, in a cell-based system, mutations in the ChEL domain impaired folding of thyroglobulin region I-II-III. Truncated thyroglobulin devoid of the ChEL domain was incompetent for cellular export; however, a recombinant ChEL protein (“secretory ChEL”) was secreted efficiently. Coexpression of secretory ChEL with truncated thyroglobulin increased intracellular folding, promoted oxidative maturation, and facilitated secretion of region I-II-III, indicating that the ChEL domain may function as an intramolecular chaperone. Additionally, we found that the I-II-III peptide was cosecreted and physically associated with secretory ChEL. A functional ChEL domain engineered to be retained intracellularly triggered oxidative maturation of I-II-III but coretained I-II-III, indicating that the ChEL domain may also function as a molecular escort. These insights into the role of the ChEL domain may represent potential therapeutic targets in the treatment of congenital hypothyroidism.

Authors

Jaemin Lee, Bruno Di Jeso, Peter Arvan

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Figure 4

Efficient exit of the isolated Tg ChEL domain from the ER.

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Secreted I-II-III protein is physically associated with secretory ChEL p...
293 cells were transiently transfected with a plasmid encoding the wild-type mouse Tg ChEL domain preceded by the prolactin signal peptide (Secretory ChEL) or were untransfected (293 control). Cells were pulse labeled for 30 minutes with 35S-labeled amino acids and chased for 0 or 4 hours as indicated, at which time the cells were lysed and both lysates and media immunoprecipitated with a rabbit polyclonal anti-Tg. Immunoprecipitates from transfected cells were divided in 2 equal portions and either mock digested or digested with endoglycosidase H (Endo H). Finally, all samples were analyzed by reducing 5.5% SDS-PAGE and fluorography. The band shift observed after digestion of secretory ChEL from the 0 chase time (shift down, lane 2) is indicative of endoglycosidase H sensitivity and defines ChEL that has not yet reached the Golgi complex; in contrast, none of the secreted ChEL shows the same endoglycosidase H sensitivity, indicating intracellular transport via the Golgi complex. The lanes shown were all run on the same gel, although they are presented noncontiguously. The position of the 76-kDa molecular mass standard is shown at left.